Streptococci
The Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, including, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid. Since its isolation more than 100 years ago, Streptococcus pneumoniae has been one of the more intensively studied microbes. For example, much of our early understanding that DNA is, in fact, the genetic material was predicated on the work of Griffith and of Avery, Macleod and McCarty using this microbe. Despite the vast amount of research with S. pneumoniae, many questions concerning the virulence of this microbe remain. It is particularly preferred to employ Streptococcal genes and gene products as targets for the development of antibiotics.
Cell surface proteins
Several cell surface associated proteins of the Staphylococci and Streptococci involved in microbial adhesion to different host tissues and considered to be important factors in bacterial pathogenesis have been identified in the last decade (see Patti, J. M., et al., MSCRAMM-Mediated Adherence of Microorganisms to Host Tissues (1994) Annu. Rev. Microbiol. 48:85-617).
Different approaches have been put forward to address such proteins from Staphylococcus aureus as antibacterial targets, e.g. fibronectin binding proteins (EP0294349, EP0397633, WO94/18327), fibrinogen binding protein (WO94/06830), collagen binding protein (WO92/07002) and bone sialoprotein binding protein (WO94/13310). The binding proteins or binding fragments thereof are used as antibacterial agents to block binding of the organism to host tissue, as vaccines to raise antibodies to the organism in the host animal or as antigens to raise therapeutic antibodies which can be used to block binding of the organism to host tissue.
Leader Peptidases
The majority of proteins that are translocated across one or more membranes from the site of synthesis are initially synthesized with an N-terminal extension known as a signal, or leader, peptide (Wickner, W., et al, (1991). Ann. Rev. Biochem. 60:101-124). Proteolytic cleavage of the signal sequence to yield the mature protein occurs during, or shortly after, the translocation event and is catalyzed in both prokaryotes and eukaryotes by enzymes known as signal, or leader, peptidases (SPases). The bacterial SPases are membrane proteins consisting of a single polypeptide anchored to the membrane by one (Gram-positive (G.sup.+) and Gram-negative (G.sup.-) bacteria) or two (G.sup.- bacteria) transmembrane sections. Predicted amino acid sequences of bacterial SPases show a high level of similarity and are known for Escherichia coli (Wolfe, P. B, et al, (1983) J. Biol. Chem. 258:12073-12080), Pseudomonas fluorescens (Black, M. T., et al, (1992). Biochem. J. 282:539-543), Salmonella typhimurium (van Dijl, J. M., et al, (1990). Mol. Gen. Genet. 223:233-240), Haemophilus influenzae (Fleischmann, R. D., et al, (1995). Science 269:496-512), Phormidium laminosum (Packer, J. C., et al, (1995). Plant Mol. Biol. 27:199-204. K. Cregg, et al: Signal peptidase from Staphylococcus aureus Manuscript JB765-96), Bradyrhizobium japonicum (Muller, P., et al, (1995). Mol. Microbiol. 18:831-840), Rhodobacter capsulatus (Klug, G., et al, (1996). GenBank entry, accession number 268305), Bacillus subtilis (two chromosomal and two of plasmid origin (Akagawa, et al, (1995) Microbiol. 141:3241-3245; Meljer, W. J. J., et al, (1995). Mol. Microbiol. 17:621-631; van Dijl, J. M., et al, (1992). EMBO J. II:2819-2828), Bacillus licheniformis (Hoang, V., et al, (1993). Sequence P42668 submitted to emb1/genbank/ddbj data banks.), Bacillus caldotyricus (van Dijl, J. M. (1993). Sequence. p41027, submitted to emb1/genbank/ddbj data banks), Bacillus amyloliquifaciens (two chromosomal genes) (Hoang, V. and J. Hofemeister. (1995). Biochim. Biophys. Acta 1269:64-68; van Dijl, J. M. (1993). Sequence p41026, submitted to emb1/genbank/ddbj data banks) and a partial sequence has been reported for Bacillus pumilis (Hoang, V. and J. Hofemeister. (1995). Biochim. Biophys. Acta 1269:64-68). These enzymes have been collectively defined as type-1 signal pepidases (van Dijl, J. M., et al, (1992). EMBO J. II:2819-2828). Although the amino acid sequences of fifteen bacterial SPases (and a sixteenth partial sequence) have now been reported, the best studied examples are leader peptidase (LPase or LepB) from E. coli and a SPase from B. subtilis (SipS).
It has been demonstrated that LPase activity is essential for cell growth in E. coli. Experiments whereby expression of the lepB gene, encoding LPase, was regulated either by a controllable ara promoter (Dalbey, R. E. and Wickner. 260:15925-15931) or by partial deletion of the natural promoter (Date, T. (1983). J. Bacteriol. 154:76-83) indicated that minimization of LPase production was associated with cessation of cell growth and division. In addition, an E. coli strain possessing a mutated lepB gene (E. coli IT41) has been shown to have a drastically reduced growth rate and display a rapid and pronounced accumulation of preproteins when the temperature of the growth medium is elevated to 42.degree. C. (Inada, T., et al, (1988). J. Bacteriol. 171:585-587). These results infer that there is no other gene product in E. coli that can substitute for LPase and that lepB is a single-copy gene in the E. coli chromosome. This is in contrast to at least two species within the G.sup.+ Bacillus genus, B. subtilis and B. amyloliquifaciens. It is known that there are at least two homologous SPase genes in each of these Bacillus species. The sipS gene can be deleted from the chromosome of B. subtilis 168 without affecting cell growth rate or viability under laboratory conditions to yield a mutant strain that can still process pre.alpha.-amylase (K. M. Cregg and M. T. Black, unpublished). A putative SPase sequence (Akagawa, et al, (1995) Microbiol. 141:3241-3245) may be the gene-product responsible for this activity and/or B. subtilis may harbor more than two SPase genes. Two or more genes encoding distinct SPase homologues reside on the chromosome of the closely related species B. amyloliquifaciens (Hoang, V. and J. Hofemeister. (1995). Biochim. Biophys. Acta 1269:64-68) and there is evidence to suggest that B. Japonicum may possess more than one SPase (Muller, P., et al, (1995). Mol. Microbiol. 18:831-840; Muller, P., et al, (1995). Planta 197:163-175). Although SPase sequences from seven genera of G.sup.+ bacteria are now known, only the single Bacillus genus amongst the G.sup.+ eubacteria has been investigated with respect to SPase characteristics. It was therefore considered of interest to determine whether a G.sup.+ eubacterium that, unlike B. subtilis and B. amyloliquifaciens, is not known for exceptional secretion activity has genes encoding more than one SPase with overlapping substrate specificities or whether it resembles E. coli and H. influenzae (and possibly other G.sup.- eubacteria)more closely in that it has a single SPase gene. The recent publication of the entire genomic sequence of the obligate G.sup.+ -like intracellular bacterium Mycoplasma genitalium also reveals an interesting feature relating to heterogeneity amongst SPases (Fraser, C. M., et al, (1995). Science 270:397-403). Inhibitors of E. coli LPase have been reported (Allsop, A. E., et al, 1995. Bioorg, & Med. Chem. Letts. 5:443-448).
Evidence has accumulated to suggest that LPase belongs to a new class of serine protease that does not utilize a histamine as a catalytic base (Black, M. T., et al, (1992). Biochem. J. 282:539-543; Sung, M. and R. E. Dalbey. (1992). J. Biol. Chem 267:13154-13159) but may instead employ a lysine side-chain to fulfill this role (Black, M. T. (1993). J. Bacteriol. 175:4957-4961; Tschantz, W. R., et al, (1993) J. Biol. Chem. 268:27349-27354). These observations and comparisons with Lex A from E. coli led to the proposal that a serine-lysine catalytic dyad, similar to that thought to operate during peptide bond hydrolysis catalyzed by LexA (Slilaty, S. N. and J. Little. (1987). Proc. Natl. Acad. Sci. USA 84:3987-3991), may operate in LPase (Black, M. T. (1993). J. Bacteriol. 175:4957-4961). Similar observations have since been made for SPase from B. subtilis (van Dijl, J. M., et al, (1995). J. Biol. Chem. 270:3611-3618) and for the Tsp periplasmic protease from E. coli (Keiler, K. C. and R. T. Sauer. (1995). Biol. Chem. 270:28864-28868); the similarities of SipS to LexA have been suggested to extend to several regions of primary structure (van Dijl, J. M., et al, (1995). J. Biol. Chem. 270:3611-3618). The serine and lysine residues (90 and 145 respectively in E. coli LPase numbering) known to be critical for catalytic activity in both E. coli LPase (Black, M. T. (1993). J. Bacteriol. 175:4957-4961; Tschantz, W. R., et al, (1993) J. Biol. Chem. 268:27349-27354) and B. subtilis SPase (van Dijl, J. M., et al, (1995). J. Biol. Chem. 270:3611-3618) and thought to form a catalytic dyad are both conserved in the S. aureus protein SpsB (S36 and K77). In addition, the aspartate at position 155 (280 in E. coli LPase numbering) is also conserved (this residue appears important for activity of the SipS SPase (van Dijl, J. M., et al, (1995). J. Biol. Chem. 270:3611-3618) but less so for LPase from E. coli (Sung, M. and R. E. Dalbey. (1992). J. Biol. Chem 267:13154-13159).
Clearly, there is a need for factors that may be used to screen compounds for antibiotic activity and which may also be used to determine their roles in pathogenesis of infection, dysfunction and disease. There is a need, therefore, for identification and characterization of such factors which can play a role in preventing, ameliorating or correcting infections, dysfunctions or diseases.
Certain embodiments of the polypeptide of the present invention has the conserved amino acid residues 34-43 and 75-84, and have amino acid sequence homology to known serine protease proteins.
References of the Background of the Invention and the Examples
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